Protein phosphorylation is a decisively important biochemical step within biological processes like: signal-transduction, membrane transport or muscle contraction. The phosphorylation is accomplished by chemical binding of a phosphate moiety to a hydroxy-amino-acid (like tyrosine or serine) of the protein chain. Phosphorylation modifies the structure and biological activity of a protein, usually in a reversible manner since the decoupling of the phosphate moiety reforms the original structure.
Specific promoters of protein phosphorylation are the protein-kinases (PK) which transfer a phosphate group from adenosine-triphosphate (ATP) to the protein substrate. Protein-phosphatases (PP) act in the contrary by splitting off the protein-bonded phosphate group. These converse functions of protein-kinases and phosphatases balances and regulates several complex cellular processes such as signal-transduction and membrane-transport.
Dysfunction of certain protein-phosphorylation processes and of the connected signalling cascades have been identified as crucial factors in several diseases. Purposeful modulation of reversible phosphorylation processes is considered therefore a promising approach to find novel therapeutics for diseases like cancer.
This concept is confirmed by recent achievements in tumour therapy with monoclonal antibodies which block the tumour-growth-promoting protein-kinases HER-2 and HER-3 in breast cancer. This tumour-suppressive action of the antibody protein is however limited in time while the host organism identifies it as antigenic protein and generates anti-antibodies to neutralize it. Providing non-antigenic drug substances for the targeted suppression or modulation of certain protein kinases/phosphatases remains an unmet medical need.
Members of the P-type ATPase (adenosine-triphosphatase) super-family are structurally related proteins involved in transport across biological membranes using energy resulting from the hydrolysis of adenosine triphosphate (ATP). According to their function as trans-membrane “ion transporters” these ATPases are classified as membrane “ion pumps”. Actually, each pumping cycle involves a phosphorylation and subsequent de-phosphorylation of the ATPase protein.
Several diseases are connected with the defective function of a certain ATPase pump, which suggests to explore the therapeutic potential of drug substances inhibiting or modulating this enzyme. Despite considerable progress in structural elucidation of ATPases, their mechanism of action and their modulation by drug substances is not fully elucidated.
One typical P-type ATPase is the Na,K-ATPase or sodium pump, which controls ionic homeostasis as well as a broad spectrum of cellular functions such as: membrane potential, pH, temperature or water osmosis. The sodium pump is involved in the regulation of important physiological processes like: muscle contraction, nervous signal transmission, renal sodium retention or vascular tone. Severe dysfunctions of the sodium pump are decisively involved in several pathologies like: essential hypertension or cardiac failure.
Prior art inhibitors of the Na,K-ATPase are the cardiac steroids of herbal origin applied since a long time for the treatment of cardiac insufficiency. However their high toxicity with lethal dose LD50 in the domain of 0.1-0.25 mg/kg body mass in human, narrows considerably their therapeutic dose range. Thus, for the cardiotonic steroid Digoxin the daily administration of 4-5 mg/patient is well tolerated but, 8-10 mg/patient/day may cause fatal toxicity. Finding of non-toxic sodium pump inhibitors is of major therapeutic interest in the treatment of cardiac insufficiency, essential hypertension and related diseases.
Candidate non-toxic modulators of the sodium pump could be the putative endogenous digitalis-like factors (EDLFs). Their existence is supported by a consistent body of experimental data but the structure of EDLFs was up to now not disclosed. Recent data demonstrated convincingly the role of Na,K-ATPase as a signalling transducer at the level of cell membranes, which suggest a novel field of potential therapeutic applications of the sodium pump modulating agents (Xie, Z., Askhari, A.; Eur. J. Biochem. 2002, 269, 2434-2439).
H+/K-ATPase or proton pump is another member of the ATPase family which transports a hydrogen ion (H+) from the cytoplasm in exchange for one potassium ion (K+) retrieved from the gastric lumen. Proton pump inhibitors (PPI) that directly bind to and inactivate the H+/K+ ATPase are disclosed in the prior art as therapeutics to treat gastric hyperacidity e.g.: omeprazole, esomeprazole, lansoprazole, pantoprazole and rabeprazole (U.S. Pat. No. 5,232,706). However, chronic administration of these PPI drugs produces side effects like: constipation, cough, dizziness or back pain. Increased susceptibility to bacterial infection due to the enhanced pH value (>4) is a further side effect caused by chronic use of synthetic proton pump inhibitory drugs.
Calcium homeostasis inside eukaryotic cells is maintained by ubiquitously distributed Ca-ATPase enzymes known as Ca pumps. Plasma membrane Ca-ATPase generally counteracts the influx of free Ca2+ ions through calcium channels and thus exerts an essential role in controlling enzymatic reactions and a broad spectrum of intracellular signalling processes. In muscle cells, the Ca-ATPase pumps back Ca2+ ions into the sarcoplasmic reticulum SR which stores Ca during muscle relaxation. This data strongly suggests that Ca-pump-modulating substances could have therapeutic applications e.g. in muscle contraction pathologies, but prior art inhibitors like thapsigargin are of limited applicability due to their advanced cellular toxicity.
Several vanadium compounds were identified as prior art inhibitors of the Na,K-ATPase, H/K-ATPase, Ca-ATPase and of other P-type ATPase enzymes. Most frequently applied are: meta-vanadate (VO3)n− or decavanadates [V10O28]6− which inhibit ATPases with IC50 (half-inhibitory concentration) values in the micro- and sub-micro-molar range but the results are not reproducible due to instant structural modification of vanadates. Decavanadate, considered the V oligomer of biochemical relevance is not stable at physiologic pH but, once formed, its disintegration is slow enough to allow the study of its effects. Despite their questionable structures, vanadates or their peroxidated derivatives the “pervanadates” have extended laboratory applications due to their very efficient inhibition of protein phosphatases which is of primary importance in investigating the complementary kinases.
Vanadate ions mimic surprisingly the rapid actions of insulin in various cell types. When administered orally to hyperglycaemic rats, vanadate stimulates glucose uptake and metabolism, and leads to normo-glycemic states. In addition, vanadate restores tissue responsiveness to insulin and hepatic glycogen levels as well as activates new synthesis of key enzymes for carbohydrate metabolism. Clinical benefits of vanadium compounds in the therapy of diabetes have been confirmed but only by short time administration in human.
Despite this emerging interest for the insulin-mimetic use of the vanadium compounds in diabetes, the toxicology of vanadium derivatives causes concern. Gastrointestinal disturbances were reported as a common toxic effect in humans and animal experiments with higher doses revealed severe signs of renal and hepatic toxicity. Long-term use of vanadium is a major concern due its progressive tissue accumulation mostly in kidney, spleen, testes, liver and bone and the production of toxic effects. The toxicity depends on the oxidation state and coordination geometry of vanadium in the order: pervanadate>vanadates>vanadyl as well as on the mode of administration [Domingo, J. L.: Vanadium and Diabetes. What about vanadium toxicity?” Mol. Cell. Biochemistry 2000, 203, 185-187].
The ABC (ATP binding-cassette) transporter proteins form an important class of membrane proteins associated with multiple cellular functions, including the elimination of xenobiotics. Although these ATP-driven efflux pumps are essential in the homeostasis of normal cells, their activity is less desired for instance by the chemo-therapy of cancer patients. This is because a survival strategy of cancer cells is to over-express ABC type multidrug-efflux pumps (MDR) making the tumours resistant to cancer drugs. Applying the cytostatic drug in greater extent is only a transient solution since it will intensify dramatically the toxic side effects. Selective inhibition of certain ATPase driven multi drug efflux pumps could provide important benefits in the cancer therapy but prior art MDR inhibitors are less efficient.
Object of the present invention is to provide new pharmaceutically active substances which are useful for prophylaxis, diagnosis and treatment of various diseases such as hypertension, diabetes, bone diseases, cardiovascular diseases, neurodegenerative pathologies and diseases, cancer, hyperacidity, osteoporosis, dental calculus, Alzheimer disease, Creutzfeld-Jakob disease and wound healing.
This object is solved by the teaching of the independent claims. Preferred embodiments are disclosed in the description, the dependent claims, the figures and the examples.